PLOS Article Microbiology

PLOS ONE
RESEARCH ARTICLE
Proteomics of extracellular vesicles produced

by Granulicatella adiacens, which causes
infective endocarditis
Sarah A. Alkandari, Radhika G. Bhardwaj, Arjuna Ellepola, Maribasappa Karched ID*
Oral Microbiology Research Laboratory, Department of Bioclinical Sciences, Faculty of Dentistry, Health
Sciences Center, Kuwait University, Kuwait, Kuwait
* mkarched@hsc.edu.kw
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a1111111111 Abstract
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a1111111111 When oral bacteria accidentally enter the bloodstream due to transient tissue damage dur-
ing dental procedures, they have the potential to attach to the endocardium or an equivalent
surface of an indwelling prosthesis and cause infection. Many bacterial species produce
extracellular vesicles (EVs) as part of normal physiology, but also use it as a virulence strat-
OPEN ACCESS
egy. In this study, it was hypothesized that Granulicatella adiacens produce EVs that possi-
bly help it in virulence. Therefore, the objectives were to isolate and characterize EVs
Citation: Alkandari SA, Bhardwaj RG, Ellepola A,
Karched M (2020) Proteomics of extracellular produced by G. adiacens and to investigate its immune-stimulatory effects. The reference
vesicles produced by Granulicatella adiacens, strain G. adiacens CCUG 27809 was cultured on chocolate blood agar for 2 days. From sub-
which causes infective endocarditis. PLoS ONE sequent broth culture, the EVs were isolated using differential centrifugation and filtration
15(11): e0227657. https://doi.org/10.1371/journal.
protocol and then observed using scanning electron microscopy. Proteins in the vesicle
pone.0227657
preparation were identified by nano LC-ESI-MS/MS. The EVs proteome was analyzed and
Editor: Jon M. Jacobs, Pacific Northwest National
characterized using different bioinformatics tools. The immune-stimulatory effect of the EVs
Laboratory, UNITED STATES
was studied via ELISA quantification of IL-8, IL-1β and CCL5, major proinflammatory cyto-
Received: December 19, 2019
kines, produced from stimulated human PBMCs. It was revealed that G. adiacens produced
Accepted: October 16, 2020 EVs, ranging in diameter from 30 to 250 nm. Overall, G. adiacens EVs contained 112 pro-
Published: November 20, 2020 teins. The proteome consists of several ribosomal proteins, DNA associated proteins, bind-
Copyright: © 2020 Alkandari et al. This is an open ing proteins, and metabolic enzymes. It was also shown that these EVs carry putative
access article distributed under the terms of the virulence factors including moonlighting proteins. These EVs were able to induce the pro-
Creative Commons Attribution License, which duction of IL-8, IL-1β and CCL5 from human PBMCs. Further functional characterization of
permits unrestricted use, distribution, and
the G. adiacens EVs may provide new insights into virulence mechanisms of this important
reproduction in any medium, provided the original
author and source are credited. but less studied oral bacterial species.
Data Availability Statement: Mass spectrometry

data is deposited in the PRIDE archive under
accession number PXD021414 and publicly
accessible via the following URL: https://www.ebi. Introduction
ac.uk/pride/archive/projects/PXD021414.
Granulicatella species, formerly known as nutritionally variant streptococci based on their
Funding: MK received funding from Kuwait
characteristic dependence on pyridoxal or cysteine supplementation for their growth in stan-
University Grant Number SRUL 01/14. The funders
had no role in study design, data collection and
dard media [1], are catalase and oxidase negative, non-motile, non-spore-forming, faculta-
analysis, decision to publish, or preparation of the tively anaerobic Gram-positive cocci [2, 3]. They are part of the normal oral flora [4], but
manuscript. cause serious infections such as infective endocarditis. The genus Granulicatella consists of 3
PLOS ONE | https://doi.org/10.1371/journal.pone.0227657 November 20, 2020 1 / 19

PLOS ONE Granulicatella estracellular vesicle proteome analysis
Competing interests: The authors have declared species: Granulicatella adiacens, Granulicatella elegans and Granulicatella balaenopterae [3].
that no competing interests exist. The species G. balaenopterae has not been isolated from human samples, whereas both G.
adiacens and G. elegans have been reported from IE cases [5, 6]. In addition, these oral com-
mensal cocci have been associated with endodontic infections [7, 8], dental caries [9], and peri-
odontitis [8, 10] via DNA-based studies. Although this association does not substantiate the
role of Granulicatella species in dental diseases, the fact that these species are causative agents
in infective endocarditis implies that they might exert similar pathogenic potential also in the
oral cavity.
Many bacterial species routinely produce extracellular vesicles (EVs) during normal growth
[11]. Gram-negative bacteria are commonly found to produce such vesicles, which are derived
from blebbing of the outer membrane and thus are called outer membrane vesicles (OMVs)
[11]. Generally, these OMVs contain outer membrane proteins, lipopolysaccharides, glycero-
phospholipids in addition to enclosed periplasmic components and bacterial nucleic acids
[11–13]. The study of the EVs was initially limited to Gram-negative bacteria, as it was thought
that the rigidity of the Gram-positive cell wall, which is rich in peptidoglycans, would not
allow vesicle blebbing [11]. However, the production of EVs was also observed in some Gram-
positive bacteria [14, 15]. Current studies [16, 17] showed that the activity of cell wall-degrad-
ing enzymes, which weaken the peptidoglycan layer and thus facilitate the release of Gram-
positive EVs, could probably explain such phenomena in Gram-positive bacteria. Similar to
Gram-negative OMVs, these EVs contain proteins, lipids, enzymes, toxins and bacterial
nucleic acids [18]. However, Gram-positive EVs can still be distinguished from OMVs as the
former lack lipopolysaccharide and enclosed periplasmic components [18].
Several studies [13, 14, 16] showed that bacteria exploit vesicle production as a virulence
strategy. Bacterial components, including virulence factors, are packed in the vesicles and
delivered to the host cells and tissues. The vesicle-derived virulence factors play an important
role in bacterial pathogenicity, e.g., by eliciting an inflammatory response, manipulating the
host’s immunity, eliminating the competing commensal microorganisms, relieving internal
stress, mediating biofilm formation, and acting as decoys absorbing and blocking cell wall-
lytic compounds and membrane-disrupting antimicrobial peptides produced by other com-
mensals and host innate immune cells [13, 14, 16]. Protein secretion in Granulicatella species
has been studied [19], but vesicle production in these species has not been investigated yet. In
this preliminary exploratory study, we isolated EVs from G. adiacens and acquired informa-
tion on the EV proteome by proteomics approach. Initial functional analyses of the EVs
showed the immunostimulatory potential against human PBMCs.
Materials and methods

Bacterial strains and culture conditions
The reference strain G. adiacens CCUG 27809 was cultured on chocolate blood agar (CBA)
with 0.001% pyridoxal hydrochloride at 37˚C and in 5% CO2 in air for 2 days. A loop-full of
colonies from the CBA plates was inoculated into brucella broth supplemented with 0.001%
pyridoxal hydrochloride and incubated as above for 2 days.
Isolation of EVs
The EVs were isolated using a previously described centrifugation and filtration protocol [20],
with slight modifications. Briefly, for pelleting the bacteria, the broth culture was centrifuged
at 5000 × g at room temperature for 10 minutes (Centrifuge 5430 R, Eppendorf AG, Ger-
many). For removing any remnants of intact bacterial cells, the supernatant was filtered
through a 0.22 μm sterile syringe filter (Millipore, Germany). The filtrate was then re-

centrifuged at 125000 × g at 4˚ C for 3 hours (Optima™ L-XP ultracentrifuge, Beckman, USA).

The obtained pellet was suspended in 300 μl sterile phosphate-buffered saline (PBS). The EVs
samples were stored at -20˚ C until used.
Preparation of whole cell protein (WCP). A loop full of colonies from the CBA plates
was suspended in 2 ml sterile PBS. The bacterial suspension was centrifuged at 5000 ×g at
room temperature for 5 minutes (Centrifuge 5430 R, Eppendorf AG, Germany). Then, after
discarding the supernatant, the pellet was washed with 2 ml sterile PBS. The bacterial whole
cell protein (WCP) was obtained by ultra-sonicating bacterial cells at 40 pulse rate on ice for 8
cycles (1 minute sonication followed by 1 minute rest per cycle) (Omni Sonic Ruptor 4000,
Omni International, USA) followed by centrifugation at 7000 ×g at 4˚ C for 10 minutes (Cen-
trifuge 5430 R, Eppendorf AG, Germany). The resulting supernatant was used as the WCP
sample and stored at -20˚ C until used.
Characterization of EVs
Scanning electron microscopy (SEM). The obtained vesicle preparations were suspended
in sterile PBS containing 3% glutaraldehyde for 2 hours on a rotator and then kept in a refrig-
erator overnight. For staining, the vesicle samples were incubated in 1% osmium tetroxide for
2 hours. For dehydration, the samples were kept in increasing concentrations of acetone from
30 to 100%, 10 minutes in each, on a rotator. The samples were then placed in a critical point
dryer for complete drying, mounted on stubs with carbon double adhesive tape and finally
coated with gold and stored in a desiccator until observation. The samples were observed on
Zeiss Leo Supra 50VP field emission scanning electron microscope (Carl Zeiss, Germany). For
comparison, SEM analysis of bacterial whole cells was also performed using the same previous
biological sample preparation protocol.
Determination of protein concentration and SDS-PAGE. Protein concentrations in the
EVs and WCP samples were determined by Quick StartTM Bradford protein microplate stan-
dard assay (Bio-Rad, USA). For protein separation, the samples were subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using the mini-PROTEAN II
cell electrophoresis system (Bio-Rad, USA). The proteins were denatured in 2× loading buffer
at 100˚C for 5 minutes, followed by centrifugation at 5000 ×g for 5 minutes. 20 μl of proteins
loaded in each well of the gel were separated on 12% SDS-PAGE at a constant 120 V. After the
run was completed, protein bands were detected using silver stain. Gel images were visualized
in G: Box Imaging System (Syngene, India). Protein banding patterns and molecular weights
of the bands were determined using GeneSys tools software.
Identification of EVs proteins by nano-LC-ESI-MS/MS. For the identification of EVs
proteins, mass spectrometry was performed by Proteome Factory (Proteome Factory AG, Ber-
lin, Germany) using nano-liquid chromatography-electrospray ionization-tandem mass spec-
trometry (nano-LC-ESI-MS/MS). With an Agilent 1100 nanoHPLC system (Agilent,
Waldbronn, Germany) interfaced to an Orbitrap Velos (Thermo Scientific, Bremen, Ger-
many) via a nanoelectrospray ion source. After pooling replicate samples from EVs prepara-
tions, proteins were reduced, alkylated and digested by trypsin (Promega, Mannheim,
Germany). Then, 400 ng of the resulting peptides were subjected to the nanoLC-ESI-MS/MS.
1% acetonitrile/0.5% formic acid was used as eluent for 5 minutes to trap and desalt the pep-
tides on the enrichment column (Zorbax 300SB-C18, 0.3 × 5 mm, Agilent). A water/acetoni-
trile (both supplemented with 0.1% formic acid) gradient from 5% to 40% acetonitrile was
then used within 120 minutes to separate the peptides on a Zorbax 300SB-C18, 75 μm x 150
mm column (Agilent). The mass spectrometer automatically recorded mass spectra, and tan-
dem mass spectra were data-dependently acquired for multiply charged ions. Protein

identification was made using the Mascot search engine (Matrix Science, London, England)
against the bacterial subset of the RefSeq protein database (National Center for Biotechnology
Information), (downloaded on 1st July 2016, 49867978 entries, NCBI, Bethesda, USA) and a
database with common protein contaminants. For MS/MS spectra where assignment of the
precursor ion’s charge state was missing, search parameters for ions from ESI-MS/MS data
acquisition was set to "2+, 3+ or 4+" according to the instrument’s and method’s standard
charge state distribution. The search parameters were: Fixed modifications: Carbamidomethyl
(C); variable modifications: Deamidated (NQ), Oxidation (M); Peptide Mass Tolerance: ±
3 ppm; Fragment Mass Tolerance: ± 0.6 Da; Missed Cleavages: 2. The inclusion criterion was:
peptides that match with a score of 20 or above. Mass spectrometry data, with the project aces-
sion number PXD021414, has been deposited at PRIDE archive (https://www.ebi.ac.uk/pride/
archive/) repository.
Bioinformatic analysis. Protein sequences from the liquid chromatography-mass spec-
trometer (LC-MS) analysis of the EVs proteome was analyzed by an in silico 2-dimensional
electrophoresis (2-DE) tool. For this, the software JVirGel, version 2.0 (http://www.jvirgel.de/
index.html), was used to obtain a theoretical (2-DE) image of the EVs proteins by uploading
protein sequences to the software [18]. The subcellular localization of the EVs proteins
detected with LC-MS/MS was predicted using the PSORTb tool, version 3.0.2 (https://www.
psort.org/psortb/) [21]. To determine if any of the secreted proteins are packed into the vesi-
cles, the prediction tool SignalP, version 5.0 (http://www.cbs.dtu.dk/services/SignalP/abstract.
php), was utilized to predict proteins secreted via the general Secretion route (Sec-pathway)
[22]. In addition to that, the prediction tool TatP (http://www.cbs.dtu.dk/services/TatP/), was
used to predict proteins secreted via the Twin-arginine translocation pathway (Tat-pathway)
[23]. To identify lipoproteins, lipoboxes were searched using the prediction tools LipoP
(http://www.cbs.dtu.dk/services/LipoP/) and PRED-LIPO (http://bioinformatics.biol.uoa.gr/
PRED-LIPO/input.jsp) [24].
Function prediction analysis. Proteins with multiple functions, known as “moonlighting
proteins”, were identified using the prediction tool moonprot, version 2.0 (http://www.
moonlightingproteins.org/) [25], and searching the database Multitask ProtDB (http://wallace.
uab.es/multitaskII/) [26]. Gene Ontology (GO) analysis of the EVs proteome was performed
using the amino acid FASTA sequences of G. adiacens. For this, GO annotations were analyzed
and plotted using the tools OmicsBox version 1.3.11 (https://www.biobam.com/download-
omicsbox/) [27], and WEGO, version 2.0 (http://wego.genomics.org.cn/) [28]. The EVs pro-
teins were grouped based on functional association networks using the tool STRING (https://
string-db.org/) [29]. Minimum interaction scores were set at a strong confidence level of 0.7.
The EVs proteins were also grouped based on different biological pathways. For this, all pro-
tein sequences from G. adiacens EVs proteome were analyzed by the Kyoto Encyclopedia of
Genes and Genome (KEGG) (https://www.genome.jp/kegg/pathway.html) pathway analysis
tool using the genus “Streptococcus” as reference [30].
Prediction of virulence factors in the EVs proteomes. To predict virulence proteins in
the EVs proteome, the tool VirulentPred (http://203.92.44.117/virulent/) [31], along with the
Virulence Factor Data Base (VFDB; http://www.mgc.ac.cn/VFs/) were used. Proteins pre-
dicted to be virulent by the previous tools were manually searched in the literature for experi-
mental evidence on their virulence properties.
Cytokine induction of human PBMCs by EVs

Isolation of human PBMCs. PBMCs from the blood of a healthy human volunteer were
isolated using Ficoll-Paque density gradient centrifugation method [32]. After obtaining

written informed consent from the donor, blood was collected by venipuncture into vacutainer
heparin tubes (3 ml per tube). The blood was then carefully layered onto 3.5 ml Ficoll-Paque
media solution (GE Healthcare, USA) in a sterile centrifugation tube. For separating mononu-
clear cells, the tubes were centrifuged at 3400 ×g at room temperature with the brakes off for
10 minutes. The layer of PBMCs, the buffy coat layer, was then transferred to another sterile
centrifugation tube. The cell isolate was washed twice by resuspending it in 5 ml RPMI
medium followed by centrifugation at 2000 rpm at room temperature with the brakes on for 5
minutes. The supernatant was discarded, and the cell pellet was finally resuspended in 1 ml
RPMI medium supplemented with 10% heat-inactivated fetal bovine serum and 2% GibcoTM
100× antibiotic-antimycotic solution. Cell concentration in the PBMCs sample was estimated
by loading 10 μl aliquot on a hemocytometer under 400× magnification.
Stimulation of human PBMCs with EVs and WCP. Isolated human PBMCs were stimu-
lated with different concentrations (10, 25, 50, and 100 μg/ml) of G. adiacens EVs and WCP
for 24 hours. For this, in a 24-well plate, 480 μl supplemented RPMI medium containing
PBMCs (106 cells per ml) was added to each well and stimulated with 20 μl of bacterial EVs or
WCP. The plate was incubated at 37˚C and in 5% CO2 in air for 24 hours. Well with 20 μl ster-
ile PBS and 480 μl RPMI medium containing PBMCs was used as negative control.
Quantitative determination of selected cytokines. The quantitative sandwich enzyme-
linked immunosorbent assay (ELISA) technique was used to quantify the production of the
human cytokines IL-8, IL-1β, and CCL5 (RANTES) from the stimulated PBMCs. For this,
ELISA immunoassay kits (Quantikine1 ELISA R&D systems, Bio-Techne, USA) were used
according to the manufacturer’s instructions. Briefly, standards, samples, and controls were
added to the wells of a 96-well microplate pre-coated with a monoclonal antibody specific for
the cytokine of interest. To allow the specific cytokine in the sample to be bound by the specific
immobilized antibody, the plate was incubated at room temperature for 2 hours. To remove
any unbound substances, the wells were washed with wash buffer using ImmunoWashTM 1575
microplate washer (Bio-Rad, USA). Then, an enzyme-linked polyclonal antibody for the spe-
cific cytokine was added to each well. After an incubation period of one hour at room tempera-
ture, the wells were washed again with wash buffer to remove any unbound antibody-enzyme
reagent. A substrate solution was then added to each well, and the microplate was incubated at
room temperature for 20–30 minutes while being protected from light. To terminate the color-
ful enzyme-substrate reaction, a stop solution was added to each well. Finally, iMarkTM micro-
plate reader was used to measure the intensity of the color developed.
Statistical analysis
All experiments were repeated twice. Statistical Package for Social Sciences Software (SPSS),
version 25, was used for data analysis. Descriptive statistics were presented using
mean ± standard deviation (SD). Independent-samples T test and Mann Whitney U test were
used to analyze differences between groups. A critical probability value (P value) of < 0.05 was
used as the cut-off level for statistical significance.
Ethical considerations
This study was approved by the ethical committee of the Health Sciences Center, Kuwait Uni-
versity (DR/EC/3413), and has been carried out in full accordance with the World Medical
Association Declaration of Helsinki. The blood donor received written information about the
nature and purposes of the study and a written informed consent was obtained upon his/her
approval to participate.

Results and discussion

Isolation of EVs
It was revealed by the current study that G. adiacens produce EVs. Vesicles of varying sizes,
ranging from 30 to 250 nm in diameter, were seen in the electron micrographs. This nano-
scale range size was consistent with other bacterial EVs [14, 15]. For comparison, images of
bacterial whole cells (Fig 1A) and the vesicle preparations (Fig 1B) were captured at the same
magnification of ×10000. Vesicle shape and size could be visualized better at a higher magnifi-
cation of ×40000 (Fig 1C).
Characterization of EVs
Determination of protein concentration and SDS-PAGE. Protein concentration in the
EVs sample from G. adiacens, 1337 μg/ml, was much lower compared to its respective WCP
sample, 3102 μg/ml. Consistently, SDS-PAGE analysis revealed that the EVs preparation from
G. adiacens showed much fewer bands on gel than its respective WCP preparation (Fig 2A).
Fig 1. SEM images of G. adiacens whole cells and the EVs preparation. SEM images of bacterial whole cells (A) and the EVs preparation (B) captured at the
magnification ×10000. (C) SEM images of the EVs acquired at ×40000.
https://doi.org/10.1371/journal.pone.0227657.g001

Fig 2. Analysis of the proteome of G. adiacens EVs. (A) SDS-PAGE gel showing protein bands from EVs and WCP preparations. (B) Protein
sequences from LC-MS analysis of the vesicle proteome analyzed by an in silico 2-DE tool. Blue spots indicate proteins with transmembrane
domains.
Identification of EVs proteins by NanoLC-ESI-MS/MS. In total, 112 proteins detected

by NanoLC-ESI-MS/MS in EVs preparations of G. adiacens, were analyzed and defined as the
EVs proteome in the present study (S1 File). These numbers were within the range of proteins
identified in previous analyses of other bacterial vesicle proteomes [14, 15].
Bioinformatic analysis. In silico 2-DE analysis of the EVs proteome showed that the
molecular mass of the proteins ranged between 20.16 kDa and 91.73 kDa (Fig 2B). Most pro-
teins from G. adiacens EVs were found to be in the predicted isoelectric point (pI) range of
3.99 and 5.6 (Fig 2B). In silico 2D gel analysis is a helpful tool when proteins with transmem-
brane helices on their membrane spanning region possess hydrophobic characters and thefore
can not be separated by electrophoresis and therefore are not visible on 2D gels ☯23].
According to the PSORTb subcellular localization prediction tool analysis, G. adiacens EVs
proteome was predicted to contain 74 cytoplasmic proteins, 8 cytoplasmic membrane proteins,
and 2 cell-wall anchored proteins; whereas the localization of 25 proteins could not be pre-
dicted. As predicted in this study, the majority of EVs proteins were cytoplasmic in G. adiacens
(66%). Cytoplasmic proteins located in other bacterial vesicles have been reported in several
earlier studies [33, 34]. Existing evidence suggests that the enormous location of cytoplasmic
proteins into vesicles is due to specific sorting mechanisms, and not due to lysis of dead cells
[35]. Importantly, cytoplasmic proteins released as part of vesicles are known to function as
adhesins, contribute to biofilm matrix formation, and help bacteria in evading the immune
system [36].
As predicted in our study by the SignalP and TatP tools, secretory proteins were packed
into the EVs of G. adiacens. According to the SignalP prediction tool, 24 proteins of G. adia-
cens EVs proteome were found to contain a signal sequence where 3 proteins were Signal pep-
tide (Sec/SPI) type and 21 were Lipoprotein signal peptide (Sec/SPII) type (S2 File). The list of
proteins predicted by SignalP and LipoP included several ABC transporter proteins,

extracellular solute-binding proteins, and CHAP-domain containing proteins. The TatP pre-
diction tool showed that none of the proteins of the G. adiacens EVs proteome contained TatP
signal sequence. Both the Sec and Tat pathways are major pathways that exist in bacteria for
proteins secretion across the cytoplasmic membrane [37, 38]. The former pathway is well
known to translocate proteins in their unfolded conformation, while the latter catalyzes the
secretion of proteins that fold before their translocation [38]. It is well-established that protein
secretion is an essential strategy in the pathogenesis of bacterial infections [37]. Lipoprotein
prediction tools (Pred-Lipo, LipoP) revealed that there were 21 lipoproteins in the G. adiacens
EVs proteome.
Function prediction analysis. The present study showed that EVs from G. adiacens carry
proteins predicted to exhibit multitasking capabilities. Table 1 lists the 15 proteins from the G.
adiacens EVs proteome that were identified as “moonlighting proteins”. Major proteins pre-
dicted as multifunctional proteins were ribosomal proteins and molecular chaperones. Addi-
tionally, a glycolytic enzyme, glyceraldehyde-3-phosphate-dehydrogenase, and a few putative
virulent proteins such as transketolase and thioredoxin were also identified. Such multifunc-
tional bacterial proteins were found to play a role in the virulence of several other human path-
ogenic bacteria; e.g., Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae,
Helicobacter pylori, and Mycobacterium tuberculosis [39–41].
Fig 3 summarizes the Gene Ontology analysis of the EVs proteome. Overall, 112 of the G.
adiacens sequences were assigned with GO annotation. For G. adiacens, the proteins were
divided into 3 groups based on GO terms: 69 proteins in “biological process” group, 21 pro-
teins in the “cellular component” group, and 77 proteins in the “molecular function” group.
According to the Gene Ontology analysis conducted in the present study, most proteins in G.
adiacens EVs proteome were predicted to be involved in molecular functions, particularly cata-
lytic and binding functions, followed by biological processes, mainly metabolic and cellular
processes. It is possible that these species might utilize nutrients in the environment by using
the metabolism-mediator proteins in the EVs [42]. Only 21 proteins in the proteome were
annotated for cellular components. Similar to other bacterial EVs, G. adiacens EVs contained
several ribosomal proteins, DNA associated proteins, binding proteins, and metabolic
Table 1. Predicted moonlighting proteins from G. adiacens EVs proteome.

GI Number Protein
gi|491800925 Chaperonin GroEL
gi|491800793 Triose-phosphate isomerase
gi|491800797 Glyceraldehyde-3-phosphate dehydrogenase
gi|491797953 Molecular chaperone DnaK
gi|491800498 Glucose-6-phosphate isomerase
gi|491801148 Elongation factor Tu
gi|491798679 6-phosphofructokinase
gi|491797130 Transketolase
gi|1489647615 Pyruvate kinase
gi|259035990 Phosphoglycerate kinase
gi|491801605 50S ribosomal protein L10
gi|1489648176 NADP-dependent phosphogluconate dehydrogenase
gi|259036192 Thioredoxin
gi|1686099964 Translation superoxide dismutase
gi|259035743 Ribosomal protein L2
https://doi.org/10.1371/journal.pone.0227657.t001

Fig 3. Gene Ontology analysis of the proteome of G. adiacens EVs preparations. Gene ontology annotation was achieved using OmicsBox and
an online software “WEGO”. Protein sequences were grouped into 3 categories based on their properties and functions.
enzymes, indicating that bacterial EVs might facilitate the transfer of functional proteins [14,
16].
Fig 4 demonstrate a STRING functional protein association network analysis of G. adiacens
EVs proteome. As demonstrated in our study, G. adiacens EVs proteome formed three distinct
protein groups based on their functional associations. These groups were carbohydrate metab-
olism, ribosomal proteins, and heat shock proteins/chaperones. Components of the carbohy-
drate metabolism network were: glyceraldehyde-3-phosphate dehydrogenase,
phosphoenolpyruvate-protein phosphotransferase, glucose-6-phosphate isomerase, phospho-
glycerate kinase, Pyruvate kinase, ATP-dependent 6-phosphofructokinase, transketolase,
pyruvate dehydrogenase E1 component, and dihydrolipoamide acetyltransferase component
of pyruvate dehydrogenase complex. The ribosomal protein group consisted mainly of the
secreted ribosomal proteins: 30S ribosomal protein S20, 50S ribosomal protein L10, 30S ribo-
somal protein S5, 50S ribosomal protein L5, 50S ribosomal protein L7/L12, 30S ribosomal pro-
tein S6; Binds together with S18 to 16S ribosomal RNA, 50S ribosomal protein L11, 30S
ribosomal protein S7, 50S ribosomal protein L2, Ribosome-recycling factor, and 50S ribosomal
protein L1. The molecular chaperones (DnaK, GroL, and GrpE) and superoxide dismutase

Fig 4. Functional protein association networks of G. adiacens EVs proteome. The online tool STRING was used for grouping the EVs
proteins based on functional networks. Minimum interaction scores were set at a strong confidence level of 0.7. The three major network groups
formed are shown in dotted circles. Seven different colors link a number of nodes and represent seven types of evidence used in predicting
associations. A red line indicates the presence of fusion evidence; a green line represents neighborhood evidence; a blue line represents co-
occurrence evidence; a purple line represents experimental evidence; a yellow line represents text mining evidence; a light blue line represents
database evidence and a black line represents co-expression evidence.
formed another cluster. A growing body of literature [40, 41] has shown that a number of
enzymes involved in the glycolytic pathway as well as molecular chaperones are recognized as
moonlighting proteins and thus could play a role in the pathogenesis of bacterial infection. Of
the glycolytic enzymes detected in EVs proteomes in this study, glyceraldehyde-3-phosphate
dehydrogenase, glucose-6-phosphate isomerase, phosphoglycerate kinase, pyruvate kinase,
and ATP-dependent 6-phosphofructokinase were found to possess moonlighting properties.
These enzymes could function as transferrin receptor, cell signaling kinase, neutrophil evasion
protein, immunomodulator, plasminogen binding protein, fibrinogen binding protein, actin
binding protein, and has a role in NAD-ribosylation activity and extracellular polysaccharide

Fig 5. KEGG pathway analysis of G. adiacens EVs proteome. All protein sequences from G. adiacens vesicle
proteome were subject to KEGG pathway analysis using the genus “streptococcus” as reference.
synthesis [40]. Moreover, the molecular chaperone DnaK was found to act as a multifunctional
protein, which could stimulate CD8 lymphocyte and monocyte chemokines production, com-
pete with HIV for binding to CCR5 receptors, and bind plasminogen [40]. In addition, it was
concluded by a previous study [43] that many bacterial ribosomal proteins could function
beyond their primary role as ribosomes, integral components of protein synthesis machinery.
These proteins could also modulate different cell processes, such as transcription, regulation of
the mRNA stability, DNA repair and replication, and phage RNA replication [43]. Further-
more, the L7/L12 ribosomal protein was experimentally proven to elicit a cell-mediated
immune response in mice [44].
KEGG pathway analysis of the EVs proteome is depicted in Fig 5. Proteins belonging to car-
bohydrate metabolism and genetic information processing were found to be the most predom-
inant in G. adiacens EVs. About 29.4% of the proteins in G. adiacens EVs proteome was
predicted to be involved in the carbohydrate metabolism and 21% in genetic information pro-
cessing. Other 12%, 9% and 7.4% of proteins were involved in protein families: metabolism,
environmental information processing and protein families: genetic information processing
respectively. As predicted by the pathway tool, a few proteins were also implicated in amino
acid metabolism, lipid metabolism, glycan metabolism, and energy metabolism. Vesicles
equipped with metabolic machineries can help bacterial colonization and host cell invasion.
For example, ATP generated in vesicles might regulate the activity of virulence factors and
facilitate cell-cell communication of bacteria [45]. Overall, metabolism related proteins in the
EVs might facilitate long-term contact between the bacterium and the epithelial cells, causing
increased epithelial cell/tissue damage.
Prediction of virulence proteins in G. adiacens EVs proteome. Our study revealed that
EVs produced by G. adiacens contained proteins that were predicted to carry virulent proper-
ties. This finding overemphasizes the role of EVs in the pathogenesis of Granulicatella infec-
tions. Table 2 show the list of 26 proteins that were predicted to be virulent from EVs
proteome of G. adiacens. The major proteins with demonstrated evidence on their virulence
properties in other bacterial species were: thioredoxin [46], aminopeptidase [47], molecular
chaperones DnaK and GroES [48, 49], Superoxide dismutase [50], Glyceraldehyde-3-phos-
phate dehydrogenase [51], phosphoglycerate kinase [52], and acyl carrier protein [53]. A vast
literature on membrane vesicles has demonstrated that a number of well-known and exten-
sively studied toxins and non-toxin virulence factors are secreted via vesicles [54]. Unlike viru-
lence factors secreted in soluble form, vesicle-associated virulence factors are provided with a

Table 2. Putative virulence factors predicted in G. adiacens EVs proteome.

GI Number Protein Literature evidence
gi|491800219 CHAP domain-containing protein [57]
gi|491799853 DNA starvation/stationary phase protection protein [58]
gi|491800704 Aminopeptidase [47]
gi|491797310 Acyl carrier protein [53]
gi|259035608| peptidase, C69 family [59]
gi|1489650858| oligoendopeptidase F [60]
gi|1489651594| 30S ribosomal protein S20 [61]
gi|491798621| hypothetical protein [62]
gi|1489651148| 2-C-methyl-D-erythritol 4-phosphate [63]
gi|1489650155| phosphonate ABC transporter [64]
gi|491798879| hypothetical protein [65]
gi|1489650855| toxic anion resistance protein [66]
gi|763046713 Copper resistance protein CopC [67]
gi|1489647414| DUF1307 domain-containing protein [68, 69]
gi|1489650843| hypothetical protein D8B48_01700 [70]
gi|259035675| 3D domain protein [71]
gi|491798643| LysM peptidoglycan-binding [72]
gi|1489650906| thiol reductase thioredoxin, partial [60]
gi|491797269| extracellular solute-binding protein [73]
gi|491796985| YlbF family regulator [74]
gi|1489650842| hypothetical protein D8B48_01695 [70]
gi|491801129| 50S ribosomal protein [75]
gi|259035743| ribosomal protein L2 [76]
gi|491801123| 50S ribosomal protein L24 [77]
https://doi.org/10.1371/journal.pone.0227657.t002
unique benefit of being protected from host proteases [13]. Moreover, vesicle-virulence factors
are delivered to host cells/tissues as concentrated packages, increasing the damage level at spe-
cific target sites. Vesicle-mediated delivery of virulence factors is a widespread mechanism
across bacterial species and genera. Similar to other oral bacteria such as Aggregatibacter acti-
nomycetemcomitans [55], Kingella kingae [56] and others that are also implicated in infective
endocarditis, G. adiacens possibly use its EVs filled with numerous putative virulent proteins
in the pathogenesis of this infection.
ELISA quantification of selected cytokines produced from stimulated

human PBMCs with EVs and WCP
As shown in Fig 6, all concentrations (10, 25, 50, and 100 μg/ml) of G. adiacens EVs triggered
the production of the selected potent proinflammatory cytokines from human PBMCs as com-
pared to the controls (0 μg/ml). Our study demonstrated that G. adiacens EVs were able to
stimulate cytokine release from human PBMCs and thus could play a role in the induction of
an inflammatory response. This finding is in accordance with previous studies [11, 14, 16] that
revealed the immuno-modulatory effects of EVs in other bacteria. In the current study, EVs
from G. adiacens induced IL-8 and IL-1β, but not CCL5, in a dose-dependent manner. G. adia-
cens EVs induced the release of IL-8 and IL-1β to significantly (P < 0.05) higher levels com-
pared to WCP. These observations overemphasize the importance of bacterial vesicle

Fig 6. ELISA quantification of IL-8 (A), IL-1β (B), and CCL5 (C) production by human PBMCs stimulated with G.
adiacens EVs and WCP (10, 25, 50, and 100 μg/ml). Cytokine induction from the EVs was considered significantly
different from WCP at � p < 0.05.

production in the activation of inflammation and thus pathogenesis of bacterial infections.

The ability of bacterial vesicles to trigger host inflammatory response is a well-established phe-
nomenon. When host epithelial cells encounter or take up the vesicles, an immediate innate
immune response begins. IL-8 and IL-1β are prominent cytokines in infective endocarditis
[78], but also in oral infections [79, 80]. IL-1β has a wide range of actions mediating inflamma-
tory host response. At low concentrations, it mediates local inflammation while at high con-
centrations it possesses endocrine effects. Due to its neutrophil recruiting property, IL-8 is a
major inflammatory cytokine induced by a variety of microbial components [81, 82].
Conclusion
To the best of our knowledge, this is the first research that presented evidence for the hypothe-
sis that G. adiacens release EVs. In this preliminary exploratory study, we found that the EVs
proteome of G. adiacens was enriched with a large number of predicted putative virulence fac-
tors. The diversity of proteins in EVs suggests possible roles of these vesicles in bacterial sur-
vival, invasion, host immune modulation as well as infection, as is the case for a number of
other bacterial species. Moreover, EVs of G. adiacens were demonstrated to be potent inducers
of proinflammatory cytokines, and importantly, the EVs were significantly more potent than
the whole cell proteins in eliciting inflammatory response. These EVs may play an important
role in the activation of inflammation and thus pathogenesis of Granulicatella infections. Fur-
ther functional characterization of the G. adiacens EVs may throw more light on how this spe-
cies may utilize vesicles to orchestrate events that may lead it from being silent normal flora
species towards infection-causing ones.
Supporting information
S1 File. List of proteins identified from G. adiacens EVs preparations by LC-MS/MS.
(XLSX)
S2 File. Lists of G. adiacens EVs secretory proteins and lipoproteins predicted by the soft-
ware tools SiganlP and LipoP, respectively.
(XLSX)
S3 File. Annotated spectra for G. adiacens EVs proteins.
(PDF)
S1 Raw images. Original gel images used in Fig 2A.
(PDF)
Acknowledgments
We acknowledge the Research Sector, Kuwait University (Project SRUL 01/14) and the College
of Graduate Studies, Kuwait University. Special gratitude is given to the Research Core Facility
(SRUL 02/13), The Nanoscopy Science Center (GE01/07) and National Unit for Environmen-
tal Research and Services, (SRUL 01/13), Kuwait University. We are very grateful to Prof. Raj
Raghupathy, at the Department of Microbiology, Faculty of Medicine, Kuwait University for
kind help with the laboratory work concerning fractionation of human peripheral blood
mononuclear cells.
Author Contributions
Conceptualization: Maribasappa Karched.

Data curation: Maribasappa Karched.

Formal analysis: Maribasappa Karched.
Funding acquisition: Maribasappa Karched.
Investigation: Sarah A. Alkandari, Radhika G. Bhardwaj, Arjuna Ellepola.
Methodology: Sarah A. Alkandari, Radhika G. Bhardwaj.
Project administration: Arjuna Ellepola, Maribasappa Karched.
Software: Maribasappa Karched.
Supervision: Maribasappa Karched.
Validation: Maribasappa Karched.
Writing – original draft: Sarah A. Alkandari.
Writing – review & editing: Radhika G. Bhardwaj, Arjuna Ellepola, Maribasappa Karched.
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PLOS ONE

Proteomics of extracellular vesicles produced

Data Availability Statement: Mass spectrometry

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Materials and methods

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centrifuged at 125000 × g at 4˚ C for 3 hours (Optima™ L-XP ultracentrifuge, Beckman, USA).

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Cytokine induction of human PBMCs by EVs

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Results and discussion

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Identification of EVs proteins by NanoLC-ESI-MS/MS. In total, 112 proteins detected

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Table 1. Predicted moonlighting proteins from G. adiacens EVs proteome.

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Table 2. Putative virulence factors predicted in G. adiacens EVs proteome.

ELISA quantification of selected cytokines produced from stimulated

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production in the activation of inflammation and thus pathogenesis of bacterial infections.

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Data curation: Maribasappa Karched.

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